IL-1 receptor antagonists with enhanced inhibitory activity

ABSTRACT

DNA molecules that code for IL-1 antagonists with improved biological activity arc described. DNA molecules coding for improved IL-1 antagonists inserted into expression vectors and host cells transformed with the said vectors containing the DNA coding for improved IL-1 antagonists and a method for the production of improved IL-1 antagonists in essentially pure form are also described. Preparations that can be injected or can be administered by some other route, consisting of a pharmaceutical preparation of the said mutants, are particularly useful as drugs in the field of therapy.

The present invention relates to mutants of the IL-1 receptor antagonist(IL-1ra ), with an improved inhibitory activity compared to the wildtype antagonist, means and methods for their preparation, and their usein the therapeutic sector in all pathologies in which IL-1 is thought tobe involved.

BACKGROUND OF THE INVENTION

IL-1 (interleukin-1) is the cytokine that the body produces, in responseto infections, various kinds of attack or antigenic stimulation, toinitiate a defence reaction of the inflammatory or immune type. IL-1 isa polypeptide of approx. 17.5 kDa in its mature form, produced mainly bythe macrophages but also by epidermal, lymphoid, vascular and epithelialcells. IL-1 is one of the principal stimulating factors of both theinflammatory and immune responses and, in its circulating form, it iscapable of acting as a hormone, inducing a broad spectrum of systemicchanges at metabolic, neurological, haematological and endocrinologicallevel. Thus, IL-1 exerts an influence on mesenchymal tissue remodelling,contributing both to destructive processes and to repair processes.Furthermore, IL-1 is an activator of lymphocytes and plays a fundamentalrole in the initiation and amplification of the immune response. IL-1also possesses strong activity of the inflammatory type, for examplestimulation of the production of prostanoids and of proteases in variouscells, including chondrocytes, fibroblasts, synovial cells, and braincells. Thus, IL-1 is involved in many components of the acute-phaseresponse and is the endogenous mediator of fever (endogenous pyrogen).IL-1 can act in synergy with other cytokines, especially TNFα,significantly amplifying its inflammatory activity.

Cloning of IL-1 has led to the identification of two active forms. Thepredominant form is IL-1β, synthesized as an inactive precursor of 269amino acids (31 kDa), which is then cut by a protease to give rise tothe active mature form (corresponding to amino acids 117-269 of theprecursor). A form that occurs about a hundred times less frequently,and is generally associated with the cells, is IL1α, which has about 26%homology with IL1β, and which is also synthesized as a precursor of 271amino acids (which, however, possesses biological activity), which thengives rise to the mature form after proteolysis of the precursor. IL-1represents a special case among the cytokines (together with fibroblastgrowth factor=FGF) in that it lacks a signal peptide and so is notsecreted via the normal routes. The most abundant extracellular formtherefore consists of the mature form of IL1β, which is thus responsiblefor the majority of the biological activities of IL-1, bothimmunostimulant and inflammatory.

In view of such activities, it has been hypothesized that IL-1 mighthave a role in the pathogenesis of inflammatory and autoimmune diseases.Thus, in the vast majority of pathologies of acute and chronicinflammation and in many autoimmune pathologies, increased production ofIL-1β has been identified as one of the main factors responsible for thepathology (Dinarello C.A. Blood 77: 1, 1991).

The biological activities of IL-1 are inhibited in the presence ofspecific inhibitors. In view of IL-1's fundamental role in thepathogenesis of many autoimmune diseases and of chronic inflammatorydiseases with tissue destruction, it is suggested that inhibition ofIL-1 could be useful in the treatment of these pathologies. IL-1ra (IL-1receptor antagonist) is a cytokine that is structurally very similar toIL-1, but is synthesized with a signal peptide and secreted as matureglycosylated protein. A non-glycosylated intracellular form of IL-1ra ,with seven extra amino acids and without a signal peptide, with activitycomparable to that of secreted IL-1ra , has also been described. IL-1rais capable of binding effectively to IL-1R_(I) and much less well toIL-1R_(II). IL-1R_(I), the type I IL-1 receptor, is a receptor thatbelongs to the immunoglobulin superfamily, composed of an extracellulardomain (which has three immunoglobulin-like units bound by disulphidebridges), a transmembrane sequence that anchors the receptor to thecell, and an intracellular domain that is responsible for transmittingthe activation signal to the interior of the cell. The other IL-1receptor, IL-1R_(II), is structurally very similar to IL-1R_(I) in theextracellular and transmembrane part, but possesses practically nointracellular domain and therefore does not seem capable of transmittingthe activation signal. It is therefore hypothesized that IL-1R_(II) doesnot have the ability to activate the cells and that IL-1R_(I) is largelyresponsible for cell activation in response to IL-1 (Arend W.P. J. Clin.Invest. 88: 1445, 1991; Dinarello C.A. & Thompson R.C. Immunol. Today11: 404, 1991). IL-1R_(II) is released naturally by the cell membrane,probably through the action of a specific protease, and once it is freein the extracellular space it is able to capture circulating IL-1β andprevent it from interacting with membrane IL-1R_(I), so that itfunctions as an IL-1 inhibitor. However, the actual biological role ofIL-1R_(II), apart from capture and inhibition of IL-1 when released bythe cell in soluble form, has not yet been elucidated definitively andthere are data in various systems that suggest possible cell activationthat is dependent on IL-1R_(II) (Boraschi et al. Neuro-Immunology ofFever p. 19, 1992; Luheshi G. et al. Am. J. Physiol. 265: E585, 1993;Kent S. et al. Proc. Natl. Acad. Sci. USA 89: 9117, 1992. IL-1ra doesnot have IL-1-like biological activity, in that it occupies IL-1R_(I)without activating the cell, and in occupying the receptor it functionsas an antagonist of IL-1 activity. On account of its antagonistactivity, IL-1ra has been used successfully in experimental models ofinflammation induced by IL-1, by LPS (a bacterial endotoxin) and by livebacteria, to inhibit the inflammatory, toxic and lethal pathologiceffects of the treatments (Ohlsson K. et al. Nature 348: 550, 1990;Wakabayashi G. et al. FASEB J. 5: 338, 1991; Alexander H.R. et al. J.Exp. Med. 173: 1029, 1991; Fischer E. et al. J. Clin. Invest. 89: 1551,1992). However, in view of the extreme potency of IL-1 (which canactivate cells by occupying fewer than ten receptors/cell), the doses ofIL-1ra necessary to obtain significant therapeutic effects in vivo areextremely high. Trials in humans in septic shock have shown a marginalefficacy of IL-1ra even at extremely high doses (Fischer C. J. et al.,J.A.M.A. 271: 1836, 1994). Accordingly, it is particularly useful to beable to modify the structure of IL-1ra so as to increase its capacityfor interaction with IL-1R_(I) and so improve its therapeutic efficacy.An IL-1ra mutant having a glycine instead of asparagine in position 91has been recently disclosed (Evans R. et al. J. Biol. Chem., 11477,1995).

SUMMARY OF THE INVENTION

The present invention relates to IL-1ra mutants that can be used asdrugs in the therapeutic field, to inhibit the pathogenetic activitiesof IL-1 with increased efficacy. More particularly, the field of theinvention comprises:

mutants of IL-1ra with improved inhibitory activity, characterized inthat at least one of the two amino acid residues in positions 91 and 109of the sequence of wild type (wt) IL-1ra is replaced by a differentresidue;

DNA sequences coding for the said mutants;

expression vectors comprising the said sequences;

host microorganisms transformed with the said vectors;

methods for the production of these mutants by culturing the transformedhost microorganisms in appropriate conditions;

use of the mutants of IL-1ra for inhibiting the biological activities ofIL-1;

pharmaceutical compositions comprising a therapeutically effectiveamount of at least one mutant of IL-1ra , a carrier and/or apharmacologically acceptable solvent, which can be used for inhibitingthe activities of IL-1, especially in situations where IL-1 could beinvolved in the pathological process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a map of expression plasmid pRSETA-IL1ra.

FIG. 2 shows a map of expression plasmid pSM441.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to IL-1ra mutants and to pharmaceuticalpreparations containing them, as active principle for the therapeuticuse as an IL-1 antagonist to inhibit the activities of IL-1 in vivo. Themutants of the invention have potential uses in the treatment oftumours, inflammatory and autoimmune diseases of the lung and airways,CNS, kidney, joints, endocardium, pericardium, eyes, ears, skin,gastrointestinal tract, urogenital system, in septic shock, bone andcartilage resorption, rheumatoid arthritis, atherosclerosis and otherchronic inflammatory pathologies with or without autoimmune involvement.

The mutants of the invention are characterized by the replacement of theamino acid residue in position 91 with an amino acid residue selectedfrom glutamine, arginine, lysine, histidine and tyrosine and/or inposition 109 with an amino acid residue selected from serine, alanine,phenylalanine, valine, leucine, isoleucine, methionine. Said mutants areprepared by means of molecular techniques of mutagenesis, to producepolypeptides that contain the sequence of IL-1ra , modified so as toimprove its capacity for interacting with IL-1R, and therefore enhanceits capacity as IL-1 antagonist. Table 1 shows the nucleotide and aminoacid sequence of IL-1ra (Sequence ID No. 1 and 2, respectively). Table 2shows the amino acid sequence of IL-1ra with indication of the selectedmutations.

According to the present invention, after transformation of hostmicroorganisms with expression vectors containing the sequences codingfor the said mutants and culturing the said organisms, polypeptides withimproved IL-1ra activity are obtained and purified, replacing theresidue in position 91 and/or the residue in position 109 in thesequence of extracellular IL-1ra or of other members of the IL-1rafamily by the above mentioned amino acid.

Preferably, the residue in position 91 is replaced by an arginineresidue and the residue in position 109 by an alanine residue. Aparticularly preferred mutant has an arginine residue in position 91 andan alanine residue in position 109.

Example 2 describes the preparation of the improved mutants of IL-1raaccording to the invention, denoted for conciseness by the followingsymbols:

MILRA-1 N91→R

MILRA-2 T109→A

MILRA-3 N91/T109→R/A

These improved mutants possess an enhanced capacity for binding to thetype I IL-1 receptor (IL-1R_(I)), together with an enhanced capacity forbinding to the type II IL-1 receptor (IL-1R_(II)), so as to provideincreased efficacy of inhibition even in pathological situations whereIL-1ra functions little owing to the possible functional involvement ofIL-1R_(II) (neutrophilia, bone resorption, CNS effects, etc.).

To construct improved mutants of IL-1ra, the cDNA of IL-1ra was clonedin a suitable vector that would permit the appropriate geneticmanipulations. The nucleotide sequence coding for IL-1ra is shown inTable 1 (Sequence ID No. 1), including the sequence that codes for thesignal peptide of 25 amino acids. The amino acid sequence of IL-1ra isshown in Tables 1 and 2 (Sequence ID No. 2). Natural IL-1ra is expressedas a protein of 177 amino acids which, after removal of the signalsequence, gives rise to a mature molecule of 152 amino acids. The cDNAof IL-1ra is used for production of the IL-1ra protein, employingstandard techniques. The gene can be inserted into an expression vectorand the expression vector can be used for transforming a suitable host.The transformed host can be cultured in conditions that favourexpression of the IL-1ra gene and, consequently, production of theIL-1ra protein.

These substitutions at positions 91 and/or 109 can be effected bysite-specific mutagenesis using appropriate synthetic oligonucleotides.The recombinant gene is then inserted into the expression vector toproduce the improved mutant.

For the therapeutic uses mentioned above, the mutated proteins of theinvention will be administered in the form of pharmaceuticalcompositions suitable for parenteral, oral or topical administration, asdescribed for example in "Remington's Pharmaceutical Sciences Handbook",Mack, Pub. Co., NY, USA, 17th ed. The average doses can vary from 2μg/kg/h to 2 mg/kg/h by intravenous infusion, and from 2 μg/kg/day to 2mg/kg/day by other administration routes.

EXAMPLE 1a Cloning of the IL-1ra gene in E. coli

The CDNA coding for the IL-1ra protein is for example isolated by PCRfrom a cDNA pool prepared by conventional techniques from cells ofmonocytemacrophage origin. The oligonucleotides that can be used forselective amplification of the cDNA coding for IL-1ra are shown:

    IL-1ra forward:  5'--GATCATATGCGACCCTCTGGGAGAAAATCC--3' (Sequence ID No.    3)                             NdeI    IL-1ra reverse:  5'--GATCTGCAGCTACTCGTCCTCCTGGAAG--3'   (Sequence ID No.    4)                             PstI

The forward oligonucleotide is designed so as to insert the NdeIrestriction site immediately upstream from the codon encoding the firstamino acid of the mature form of the protein (R26). The NdeI sitepermits insertion of a non-natural methionine which will constitute theinitial amino acid of the recombinant proteins in question. Similarly,by means of the reverse oligonucleotide, a PstI restriction site isinserted immediately downstream from the stop codon of the protein. Theamplified fragment is cloned at the NdeI and PstI sites of theexpression vector pRSETA, obtaining the plasmid pRSETA-IL1ra. The map ofthe plasmid pRSETA-IL1ra is shown in FIG. 1.

EXAMPLE 1b Clonina of the IL-1ra gene in B. subtilis

The cDNA coding for the IL-1ra protein is for example isolated by PCRfrom a cDNA pool, prepared by conventional techniques from cells ofmonocytemacrophage origin. The oligonucleotides that can be used forselective amplification of the cDNA encoding IL-1ra are shown:

    IL-1ra forward:    5'-GGGAATTCTTATGCGACCCTCTGGGAGAAAATCC-3'             (Sequence ID No. 5)         EcoRI    IL-1ra reverse:  5'--GGCTGCAGCTACTCGTCCTCCTGGAAG--3' (Sequence ID No. 6)                           PstI

The forward oligonucleotide has been designed so as to insert the EcoRIrestriction site immediately upstream from the codon encoding the aminoacid methionine (indispensable for directing the start of translation ofmRNA), followed by the codon encoding the first amino acid of the matureform of the protein (R26).

Similarly, by means of the reverse oligonucleotide, a PstI restrictionsite is inserted immediately downstream from the stop codon of theprotein.

The amplified fragment is cloned at the EcoRI and PstI sites of theexpression vector pSM671, obtaining the plasmid pSM441. The map ofplasmid pSM441 is shown in FIG. 2.

EXAMPLE 2 Mutations of the IL-1ra gene

To obtain the desired mutations, part of the sequence coding for IL-1ra(amino acids 30-152) is transferred by cloning from plasmid pSM441 intothe mutagenesis plasmid Bluescript SK⁺ between the SpeI and PstIrestriction sites, obtaining the plasmid BSK-IL1ra. Mutagenesis iseffected using synthetic oligonucleotides, obtained with an AppliedBiosystems 392 oligonucleotide synthesizer and utilizing phosphoramiditechemistry.

To obtain mutagenesis at site 91 of IL-1ra, the following complementaryoligonucleotide, which is the reverse of the coding sequence, was used(Sequence ID No. 7):

    1. 5' GTC CTG CTT TCT GCG CTC GCT CAG 3'                           91

Sequence 1 can be modified on the anticodon corresponding to amino acid91 so as to code for the other amino acids in that position.

To obtain the plasmid BSK-MILRA-1 (containing the mutation in position91), the synthetic oligonucleotide is mixed with the single strand ofDNA from the plasmid BSK-IL-1ra in a pairing buffer (5 pmol ofoligonucleotide with 0.2 pmol of single strand in 10 ml of buffer), themixture is heated to 70° C., then cooled slowly to 30° C. in 40 min andfinally placed in ice. 1 ml of synthesis buffer, 1 ml (3 units) of T4bNA ligase and 1 ml (0.5 unit) of T7 DNA polymerase are added to themixture. After incubation for 1 hour at 37° C., the mixture is used totransform the competent cells. Identification of the positive clones iscarried out by DNA sequencing.

Similarly, to obtain the plasmid BSK-MILRA-2, containing the T109→Amutation, the following oligonucleotide is synthesized (Sequence ID No.8):

    2. 5' CTC AAA ACT GGC GGT GGG GCC 3'                      109

Alternatively, it is possible to use suitable oligonucleotides that codefor the other amino acids in position 109. Then the same procedure isfollowed as for the N91→R mutation.

EXAMPLE 3 Insertion of the modified IL-1ra genes into expression vectors

The mutated sequence in plasmid BSK-MILRA-1 or BSK-MILRA-2 (mutation inposition 91 or 109) is cut with SpeI and PstI and cloned directly intothe expression plasmid pRSETA-IL1ra, between the same restriction sites,obtaining the expression plasmids pT7MILRA-1 and pT7MILRA-2. Cloning ofthe mutated sequences into the expression vector pSM441 was accomplishedsimilarly, obtaining the expression plasmids pSM539 and pSM540. Thesequence of the double mutant N91→R and T109→A is obtained by cloningbetween the SpeI and PstI sites of the vector pRSETA-IL1ra or of thevector pSM441, the SpeI-HaeII fragment of the BSK-MILRA-1 clone (aminoacids 30-96) and HaeII-PstI fragment of the BSK-MILRA-2 clone (aminoacids 97-152), obtaining the clone pT7MILRA-3 and pSMILRA-3respectively.

EXAMPLE 4 Expression of the modified genes of IL-1ra

The expression plasmids pT7MILRA-1, pT7MILRA-2 and pT7MILRA-3 aretransferred independently into cells of E. coli strain BL21 (DE3), whichpossess the gene for T7 RNA polymerase and so are capable oftranscribing coding sequences downstream from plasmid T7. The cells aregrown in LB culture medium containing 100 mg/l of ampicillin until anOD_(590nm) of 0.7 is reached. Expression is induced at this point with0.4 mM IPTG for 3-4 hours. The cells containing the protein expressedare harvested by centrifugation and frozen at -80° C. until the time ofpurification.

The expression plasmids pSM539, pSM540 and pSMILRA3 are transferredindependently to cells of B. subtilis strain SMS118. The cells are grownin LB culture medium containing 5 mg/l of chloramphenicol for 16 hoursat 30-37° C. The cells containing the protein expressed are harvested bycentrifugation and frozen at -80° C. until the moment of purification.

EXAMPLE 5 Purification of the modified proteins expressed

For the mutant proteins of IL-1ra with improved activity to be obtainedin an essentially pure form, extraction from the bacteria andpurification of the homogenate are undertaken, for example according tothe procedures indicated below.

1. Extraction: The bacteria are thawed, resuspended 1:3 (wetweight:volume) in 25 mM MES pH 6.25, 1 mM EDTA (buffer A) and sonicatedin melting ice for 5 min (E. coli) or 15 min (B. subtilis) at a power of60-70 W at intervals of 30 s. The homogenate is centrifuged at 30 000×gat 4° C. (or is put through some other suitable operation, e.g.tangential filtration). Aliquots of the supernatant and sediment areanalysed in SDS-PAGE. Alternatively, extraction from the bacteria can beeffected by some other suitable method. Aliquots of the supernatant andof the sediment are analysed in SDS-PAGE.

2. Q-Sepharose FF: The supernatant is adjusted to pH 6.0-6.5 and to aconductivity of 3-5 mS/cm and batch-incubated for 3 hours at 4° C. withstirring, with 1 ml/g of cells (wet weight) of Q-Sepharose Fast Flow (orsome other suitable stationary ion-exchange phase) equilibrated inbuffer A. Alternatively, the treatment can be carried out in a column,with isocratic elution in buffer A. The unadsorbed matter is thencollected by filtration on a porous diaphragm. The gel is washed for 20min as above in 2 volumes of buffer A; the wash liquid is collected asabove. Aliquots of the unadsorbed matter and of the wash liquid areanalysed in SDS-PAGE. The mutant protein of IL-1ra is found in theunadsorbed matter and in the wash liquid.

Point 2 can be postponed and effected in a column with isocratic elutionin buffer A instead of gel filtration as in point 5.

3. S-Sepharose FF: The unadsorbed matter and the wash liquid of theQ-Sepharose FF are combined, filtered on 0.45 mm and loaded onto acolumn of S-Sepharose Fast Flow (or some other suitable stationaryion-exchange phase) equilibrated in buffer A. The unadsorbed matter iscollected and buffer A is passed through until the baseline (A₂₈₀) fallsto zero. Then a linear gradient is applied from 0.05 to 0.5M NaCl inbuffer A in 2 column volumes, collecting the fractions. Alternatively,the linear gradient can be replaced by a stepped gradient, withintervals of 0.1M NaCl. Aliquots of the unadsorbed matter, of thefractions from gradient elution and of the eluted peak are analysed inSDS-PAGE. The mutant IL-1ra protein is found in the central fractions ofthe first peak eluted between 0.2 and 0.4M NaCl. Passage throughQ-Sepharose FF and through S-Sepharose FF, as in points 2 and 3, can bereversed.

4. Filtration: The eluted peak of IL-1ra is concentrated and dialysed onMillipore Centriprep 10 filters, until the NaCl concentration fallsbelow 50 mM; high molecular weight contaminants are removed byfiltration on Millipore Centricon 100 centrifuge filters. Aliquots fromthe various filtrates are analysed in SDS-PAGE. Depending on the volumesto be treated, the operations described can be performed with varioussystems, using the same type of membrane.

5. Bio Gel P10: Any contaminants present, whether of higher or lowermolecular weight, are removed by gel filtration in a column of Bio GelP10 from Bio-Rad (or some other equivalent stationary phase for gelfiltration) equilibrated and eluted in buffer A. Aliquots of thefractions eluted are analysed in SDS-PAGE. The mutant IL-1ra protein isfound in the middle fractions of the first peak eluted after theexcluded volume peak, in essentially pure form.

EXAMPLE 6 Characterization of inhibitory activity with the assay ofbinding to IL-1R_(I) and IL-1R_(II)

The inhibitory activity of the improved IL-1ra mutants is measured inreceptor binding assays, using IL-1ra as the reference standard. Cellswhich express IL-1R_(I) selectively (e.g. the murine thymoma cloneEL4-6.1) and cells which express IL-1R_(II) selectively (e.g. Burkitt'shuman lymphoma RAJI clone 1H7) are chosen as target cells. The number ofreceptors per cell and the binding affinity (Kd) in both the lines arecalculated from saturation curves obtained by incubating the cells (10⁶cells/test tube in a final volume of 0.1 ml of culture medium with NaN₃)with increasing doses of IL-1β labelled with 125_(I) in the presence orabsence of a 500-fold molar excess of unlabelled IL-1β (for calculatingnon-specific binding, generally always less than 5-10%) for the optimumtimes and at the optimum temperatures for attaining equilibrium(generally 2 hours at room temperature) (Scapigliati et al. FEBS Lett.243: 394, 1989). To calculate the inhibition activity, tests areconducted by incubating the cells with a concentration of radiolabelledIL-1β corresponding to approximately half of the Kd in the absence or inthe presence of stepped doses of IL-1ra (reference standard) or of theimproved mutant.

The improved inhibitory activity of the mutants is determined bycalculating the shift of the inhibition curve towards the lower doseswhen compared with the curve of IL-1ra. An example of the resultsobtained (for MILRA-1 MILRA 2, and MILRA 3) is given in Table 3.

EXAMPLE 7 Characterization of antagonist activity with in-vitro assaysof inhibition of IL-1β

The antagonist activity of the improved mutants of IL-1ra is evaluatedby means of in-vitro biological assays of IL-1 activity. Two assays ofthis kind are described below.

1. Proliferation of murine thymocytes: normal thymocytes are obtained bybreaking up thymus glands of C3H/HeJ mice (resistant to bacterialendotoxin) aged 4-8 weeks. The thymocytes (5×10⁵ cells/well of Cluster⁹⁶plates) are incubated for 72 hours in RPMI-1640 culture medium withaddition of antibiotics, L-glutamine, 2-ME, HEPES and 5% foetal calfserum at 37° C. in air with 5% CO₂. Incubation takes place in thepresence of a suboptimal dose of the mitogen PHA (1.5 mg/ml, which doesnot induce significant proliferation of thymocytes) and steppedconcentrations of IL-1β. To determine the proliferation of thethymocytes induced by IL-1, at the end of 72 hours of incubation 25 mlof culture medium containing 0.5 mCi of tritiated thymidine are added toeach well. After another 18 hours, the cells from each well areharvested onto small glass-fibre disks and the radioactivityincorporated (proportional to the proliferation of the cells) ismeasured with a β-counter. For determination of the inhibitory capacityof the improved mutants of IL-1ra , the proliferation of the thymocytesis measured in response to the minimum dose of IL-1 that induces optimumproliferation (generally around 0.3 ng/ml) in the absence or in thepresence of stepped doses of IL-1ra (control standard) or of theimproved mutants. The improved inhibitory activity of the mutants isdetermined by calculating the shift of the inhibition curve towards thelower doses when compared with the curve of IL-1ra . An example of theresults obtained (for MILRA-1) is presented in Table 3.

2. Induction of IL-6: cells of the continuous line of human osteosarcomaMG-63 are incubated (5×10⁴ cells/well of Cluster⁹⁶) for 48 hours inRPMI-1640 culture medium with antibiotics, L-glutamine, HEPES and 5%foetal calf serum in the absence or in the presence of stepped doses ofIL-1β.

The quantity of IL-6 in the culture supernatants is measured by acommercial ELISA assay or determined in the biological assay ofproliferation of 7TD1 cells. For evaluation of the antagonist capacityof the improved mutants of IL-1ra , the production of IL-6 is measuredin MG-63 cells stimulated with the minimum dose of IL-1 capable ofoptimum induction of IL-6 (around 0.3 ng/ml) in the absence or in thepresence of stepped doses of IL-1ra (control standard) or of theimproved mutants of IL-1ra. The improved inhibitory activity of themutants is determined by calculating the shift of the inhibition curvetowards the lower doses when compared with the curve of IL-1ra .

An example of the results obtained (for MILRA-1 MILRA 2, anb MILRA-3) ispresented in Table 3.

EXAMPLE 8 Characterization of antagonist activity by in-vivo assays ofinhibition of IL-1β

The inhibitory activity of the improved mutants of IL-1ra is evaluatedusing in-vivo biological assays of IL-1 activity. Two assays of thiskind are described below.

1. Induction of hypoglycaemia: C3H/HeJ mice or mice of some other strain(3-5 mice/test group) receive stepped doses of IL-1β by intraperitonealadministration. After two hours the animals are killed and the blood iscollected for preparation of the serum. The serum glucose content isdetermined after reaction with glucose oxidase in a commercialcalorimetric assay. To evaluate the antagonist capacity of the improvedmutants of IL-1ra , induction of hypoglycaemia in vivo is effected byadministering the minimum dose of IL-1β capable of inducing the optimumeffect (generally around 5 mg/kg) in the absence or in the presence ofstepped doses of IL-1ra (control standard) or of the improved mutants ofIL-1ra.

The improved inhibitory activity of the mutants is determined bycalculating the shift of the inhibition curve towards the lower doseswhen compared with the curve of IL-1ra . An example of the resultsobtained (for MILRA-1a) is presented in Table 3.

2. Induction of neutrophilia: C3H/HeJ mice or mice of some other strain(3 mice/test group) receive stepped doses of IL-1β with a singleintraperitoneal administration. The increase of circulating neutrophilsinduced by IL-1 is evaluated by cytofluorimetry four hours after thetreatment. The antagonist capacity of the improved mutants of IL-1ra isevaluated as inhibition of the neutrophilia induced by the minimum doseof IL-1 necessary to obtain the optimum increase of circulatingneutrophils (generally around 150 ng/kg). IL-1ra (control standard) andthe improved mutants are administered intraperitoneally on threeoccasions, at times -15 min, 0, +15 min, relative to IL-1β. The improvedinhibitory activity of the mutants is determined by calculating theshift of the curve of inhibition towards the lower doses when comparedwith the curve of IL-1ra.

An example of the results obtained (for MILRA-1 MILRA-2, and MILRA-3) ispresented in Table 3.

                                      TABLE 1    __________________________________________________________________________    NUCLEOTIDE AND AMINO ACID SEQUENCE OF IL-1ra    __________________________________________________________________________    1  ATGGAAATCTGCAGAGGCCTCCGCAGTCACCTAATCACTCTCCTCCTCTTCCTGTTCCAT    1  M E I C R G L R S H L I T L L L F L F H    61 TCAGAGACGATCTGCCGACCCTCTGGGAGAAAATCCAGCAAGATGCAAGCCTTCAGAATC    21 S E T I C R P S G R K S S K M Q A F R I    121       TGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTG    41 W D V N Q K T F Y L R N N Q L V A G Y L    181       CAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATGTGGTACCCATTGAGCCTCATGCT    61 Q G P N V N L E E K I D V V P I E P H A    241       CTGTTCTTGGGAATCCATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATGAG    81 L F L G I H G G K M C L S C V K S G D E    301       ACCAGACTCCAGCTGGAGGCAGTTAACATCACTGACCTGAGCGAGAACAGAAAGCAGGAC    101       T R L Q L E A V N I T D L S E N R K Q D    361       AAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCACCACCAGTTTTGAGTCTGCCGCC    121       K R F A F I R S D S G P T T S F E S A A    421       TGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAAT    141       C P G W F L C T A M E A D Q P V S L T N    481       ATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACTTCCAGGAGGACGAG                                           531    161       M P D E G V M V T K F Y F Q E D E   177    __________________________________________________________________________

                  TABLE 2    ______________________________________    AMINO ACID SEQUENCE OF IL-1ra    ______________________________________                                            5    MEICR  GLRSH   LITLL     LFLFH  SEITC   RPSGR    10     15      20        25     30      35    KSSKM  QAFRI   WDVNQ     KTFYL  RNNQL   VAGYL    40     45      50        55     60      65    QGPNV  NLEEK   IDVVP     IEPHA  LFLGI   HGGKM    70     75      80        85     90      95    CLSCV  KSGDE   TRLQL     EAVNI  TDLSE   NRKQD                                            R    100    105     110       115    120     125    KRFAF  IRSDS   GPTTS     FESAA  CPGWF   LCTAM                   A    130    135     140       145    150     152    EADQP  VSLTN   MPDEG     VMVTK  FYFQE   DE    ______________________________________     NOTES:     The amino acids in italics represent the signal peptide, which is removed     in the mature protein.     The numbering refers to the mature protein, without the signal peptide.     The amino acids in bold represent the two positions where the     substitutions were planned, with the preferred substitution shown below.

                                      TABLE 3    __________________________________________________________________________    BIOLOGICAL ACTIVITY OF MILRA-1, MILRA-2 AND MILRA-3    BIOLOGICAL ASSAY    OF IL-1β INHIBITION                IL-1ra MILRA-1                              MILRA-2                                    MILRA-3    __________________________________________________________________________    In vitro:    Binding to IL-1R.sub.I *                0.34 nM                       0.17 nM                              0.19 nM                                    0.22 nM    Binding to IL-1R.sub.II *                19.60 nM                       13.20 nM                              1.80 nH                                    70.70 nM    Thymocyte proliferation**    to IL-1β 300 pg/ml                4.10 ng/ml                       1.78 ng/ml                              1.75 ng/ml                                    n.t.    to IL-1β 30 pg/ml                813 pg/ml                       n.t.   n.t.  285 pg/ml    IL-6 production**                3.60 ng/ml                       0.62 ng/ml                              1.12 ng/ml                                    n.t.    In vivo:    Hypoglycaemia***                968.0 μg/kg                       128.0 μg/kg                              n.t.  76.0 μg/kg    Neutrophilia****                225.0 μg/kg                       <22.5 μg/kg                              n.t.  n.t.    __________________________________________________________________________     *Displacement of .sup.125 I IL1β equilibrium binding 80.4-0.6 Nm     .sup.125 I IL1β on 10.sup.6 cells).     **Murine thymocyte proliferation in response to 0.3 ng/ml of IL1β.     ***Hypoglycaemia induced by in the mouse by 5.0 μg/kg IL1β.     ****Neutrophilia induced in the mouse by 150 ng/kg of IL1β.     Doses reported are ID.sub.50 except for neutrophilia, where ID.sub.100 is     indicated.     n.t. not tested

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 8    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 531 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: both              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Homo sapi - #ens    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..531    -     (ix) FEATURE:              (A) NAME/KEY: sig.sub.-- - #peptide              (B) LOCATION: 1..75    -     (ix) FEATURE:              (A) NAME/KEY: mat.sub.-- - #peptide              (B) LOCATION: 76..531    -     (ix) FEATURE:              (A) NAME/KEY: mutation              (B) LOCATION: replace(346. - #.348, "cgc")    #/note= "CGC is the codon for the preferred    #Arg amino acid substitution at this                   position."    -     (ix) FEATURE:              (A) NAME/KEY: mutation              (B) LOCATION: replace(400. - #.402, "gcc")    #/note= "GCC is the codon for the    > Ala amino acid substitution at this                   position."    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - ATG GAA ATC TGC AGA GGC CTC CGC AGT CAC CT - #A ATC ACT CTC CTC CTC      48    Met Glu Ile Cys Arg Gly Leu Arg Ser His Le - #u Ile Thr Leu Leu Leu    - #10    - TTC CTG TTC CAT TCA GAG ACG ATC TGC CGA CC - #C TCT GGG AGA AAA TCC      96    Phe Leu Phe His Ser Glu Thr Ile Cys Arg Pr - #o Ser Gly Arg Lys Ser    #1               5    - AGC AAG ATG CAA GCC TTC AGA ATC TGG GAT GT - #T AAC CAG AAG ACC TTC     144    Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Va - #l Asn Gln Lys Thr Phe    #         20    - TAT CTG AGG AAC AAC CAA CTA GTT GCT GGA TA - #C TTG CAA GGA CCA AAT     192    Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Ty - #r Leu Gln Gly Pro Asn    #     35    - GTC AAT TTA GAA GAA AAG ATA GAT GTG GTA CC - #C ATT GAG CCT CAT GCT     240    Val Asn Leu Glu Glu Lys Ile Asp Val Val Pr - #o Ile Glu Pro His Ala    # 55    - CTG TTC TTG GGA ATC CAT GGA GGG AAG ATG TG - #C CTG TCC TGT GTC AAG     288    Leu Phe Leu Gly Ile His Gly Gly Lys Met Cy - #s Leu Ser Cys Val Lys    #                 70    - TCT GGT GAT GAG ACC AGA CTC CAG CTG GAG GC - #A GTT AAC ATC ACT GAC     336    Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Al - #a Val Asn Ile Thr Asp    #             85    - CTG AGC GAG AAC AGA AAG CAG GAC AAG CGC TT - #C GCC TTC ATC CGC TCA     384    Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Ph - #e Ala Phe Ile Arg Ser    #        100    - GAC AGT GGC CCC ACC ACC AGT TTT GAG TCT GC - #C GCC TGC CCC GGT TGG     432    Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Al - #a Ala Cys Pro Gly Trp    #   115    - TTC CTC TGC ACA GCG ATG GAA GCT GAC CAG CC - #C GTC AGC CTC ACC AAT     480    Phe Leu Cys Thr Ala Met Glu Ala Asp Gln Pr - #o Val Ser Leu Thr Asn    120                 1 - #25                 1 - #30                 1 -    #35    - ATG CCT GAC GAA GGC GTC ATG GTC ACC AAA TT - #C TAC TTC CAG GAG GAC     528    Met Pro Asp Glu Gly Val Met Val Thr Lys Ph - #e Tyr Phe Gln Glu Asp    #               150    #            531    Glu    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 177 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Met Glu Ile Cys Arg Gly Leu Arg Ser His Le - #u Ile Thr Leu Leu Leu    - #10    - Phe Leu Phe His Ser Glu Thr Ile Cys Arg Pr - #o Ser Gly Arg Lys Ser    #1               5    - Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Va - #l Asn Gln Lys Thr Phe    #         20    - Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Ty - #r Leu Gln Gly Pro Asn    #     35    - Val Asn Leu Glu Glu Lys Ile Asp Val Val Pr - #o Ile Glu Pro His Ala    # 55    - Leu Phe Leu Gly Ile His Gly Gly Lys Met Cy - #s Leu Ser Cys Val Lys    #                 70    - Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Al - #a Val Asn Ile Thr Asp    #             85    - Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Ph - #e Ala Phe Ile Arg Ser    #        100    - Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Al - #a Ala Cys Pro Gly Trp    #   115    - Phe Leu Cys Thr Ala Met Glu Ala Asp Gln Pr - #o Val Ser Leu Thr Asn    120                 1 - #25                 1 - #30                 1 -    #35    - Met Pro Asp Glu Gly Val Met Val Thr Lys Ph - #e Tyr Phe Gln Glu Asp    #               150    - Glu    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -      (v) FRAGMENT TYPE: N-terminal    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Homo sapi - #ens    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #           30     CTGG GAGAAAATCC    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 28 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -      (v) FRAGMENT TYPE: C-terminal    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Homo sapi - #ens    #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #             28   TCCT CCTGGAAG    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 34 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -      (v) FRAGMENT TYPE: N-terminal    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Homo sapi - #ens    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #        34        CCCT CTGGGAGAAA ATCC    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 27 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -      (v) FRAGMENT TYPE: C-terminal    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Homo sapi - #ens    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #             27   CCTC CTGGAAG    - (2) INFORMATION FOR SEQ ID NO: 7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -      (v) FRAGMENT TYPE: internal    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Homo sapi - #ens    -     (ix) FEATURE:              (A) NAME/KEY:              (B) LOCATION: 13..15    #/note= "anticodon corresponding to                   amino aci - #d 91 of mature IL-1ra"    #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                24TCGC TCAG    - (2) INFORMATION FOR SEQ ID NO: 8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -      (v) FRAGMENT TYPE: internal    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Homo sapi - #ens    -     (ix) FEATURE:              (A) NAME/KEY:              (B) LOCATION: 10..12    #/note= "anticodon corresponding to                   amino aci - #d 109 of mature IL-1ra"    #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                GGGC C    __________________________________________________________________________

We claim:
 1. A mutant of IL-1ra in which at least one of the amino acidresidues in positions 91 and 109 of the sequence of wild type IL-1ra isreplaced by a residue selected from the group consisting of glutamine,arginine, lysine, histidine, and tyrosine for position 91 and by aresidue selected from the group consisting of serine, alanine,phenylalanine, valine, leucine, isoleucine, and methionine for position109.
 2. The mutant of IL-1ra according to claim 1, in which at least oneof the amino acid residues in positions 91 and 109 of the sequence ofwild type IL-1ra is replaced by a residue selected from the groupconsisting of arginine, lysine, histidine, and tyrosine for position 91and by a residue selected from the group consisting of alanine,phenylalanine, valine, leucine, isoleucine, and methionine for position109.
 3. The mutant of IL-1ra according to claim 1, in which the aminoacid residue in position 91 of the sequence of wild type IL-1ra isreplaced by arginine.
 4. The mutant of IL-1ra according to claim 1, inwhich the amino acid residue in position 109 of the sequence wild typeIl-1ra is replaced by alanine.
 5. The mutant of IL-1ra according toclaim 1, in which both of the amino acid residues in positions 91 and109 of the sequence of wild type IL-1ra are replaced respectively byarginine and alanine.
 6. The mutant of IL-1ra according to claim 1, inwhich the sequence of IL-1ra is the sequence of an extracellular orintracellular IL-1ra.
 7. A DNA sequence coding for the mutant of IL-1raof claim
 1. 8. The DNA sequence coding for the mutant of IL-1raaccording to claim 7, further comprising regulating elements that permittheir insertion into expression vectors.
 9. An expression vectorcontaining a DNA sequence of claim
 7. 10. The expression vector of claim9, being a plasmid selected from the group consisting of pT7MILRA-1,pT7MILRA-2, pSM539, pSM540, pT7MILRA-3, and pSMILRA-3.
 11. A host celltransformed with the expression vector as in claim
 9. 12. The host cellof claim 11, being a cell selected from the group consisting of E. colistrain BL21 and B. subtilis strain SMS118.
 13. A method for theproduction, in essentially pure form, of the mutant of IL-1ra of claim 1which comprises culturing in a culture medium host cells havingexpression vectors containing a nucleotide sequence for the mutant andrecovering an expression product from the host cells or from the culturemedium.
 14. A pharmaceutical composition containing as active principlea mutant of IL-1ra as in claim 1 mixed with a suitable vehicle.
 15. Amethod for inhibiting binding to IL-1R_(I) receptors in cells whichcomprises treating said cells with the mutant of IL-1ra of claim
 1. 16.A method for inhibiting proliferation of thymocytes which comprisestreating cells with the mutant of IL-1ra of claim
 1. 17. A method forantagonizing the production of IL-6 which comprises treating cells withthe mutant IL-1ra of claim 1.